Mapping molecular highways
Second-harmonic generation reveals polarity in neurons
Many molecular highways within neurons have a preferred direction, say researchers from Cornell University in Ithaca, N.Y. They discovered this by mapping these pathways in mouse brain tissue using second-harmonic generation. It had been thought that all such thoroughfares in dendrites – branchlike neuronal projections – had mixed polarity, with some pointing in one direction and the rest the other way.
This information on polarity could prove vital in determining how molecular motors transport cargo, said Watt W. Webb, a professor of applied physics and engineering at Cornell and leader of the group. “Understanding the microtubule track orientation of polarity is the first step in understanding this trafficking process. This is analogous to figuring out a map of the highway, or microtubule, polarities before categorizing the directionality of the cars and trucks, or the molecular motors.”
Past attempts at determining microtubule polarity used electron microscopy. This method had shown that immature neuronal axons had microtubules that were all oriented similarly, whereas mature dendrites showed mixed polarity, with about half their microtubules pointed in one direction and the others, the opposite way.
With second-harmonic-generation microscopy, polarized microtubules in organized dendritic arrays are visualized in acute brain slices. Courtesy of Alex C. Kwan and Watt W. Webb, Cornell University.
Although this technique yielded data, difficulties in applying the method successfully to intact tissues limited it usefulness. Seeking a better method, the Cornell group turned to second-harmonic generation. This is a nonlinear optical process in which two incoming photons are converted into a single outgoing photon of twice the energy and half the wavelength. It’s a coherent process, and in biological samples, it arises only from cellular elements.
Microtubules are a good choice for this approach because, as a consequence of their tubulin proteins, they’re highly polarizable. What’s more, they are aligned asymmetrically along the microtubule’s longitudinal axis. Thus, the resulting second-harmonic-generation emission is directional and at an angle that depends on the molecular structure.
To better understand what was going on, the researchers modeled microtubule optics. These simulations made it obvious that polarity is a major determinant of whether there is significant forward-directed second-harmonic generation. The group reported on its research in the Aug. 12 issue of PNAS.
Based on these and other modeling results, the researchers reasoned that the ratio of forward- to backward-directed signal would indicate the degree of polarization. A high ratio would mean the microtubules were polarized, while a low ratio would mean they weren’t.
However, measuring the ratio with accuracy wasn’t easy. “Because second-harmonic generation from microtubules is a relatively weak signal, primarily in the forward optical direction, we had to optimize its quantitative detection to improve sensitivity,” said lead author and graduate student Alex C. Kwan.
To do this, the researchers used photon counting to reduce the background signal. They also employed high-transmission, extra-narrowband emission filters from Semrock Inc. of Rochester, N.Y. Kwan noted that Chroma Technology Corp. of Rockingham, Vt., now also has such filters.
The filters in the study had a bandwidth of 10 nm, and the researchers used them to block stray autofluorescence and also infrared light from the excitation source, a Spectra-Physics Ti:sapphire laser generating 774-nm pulses. They sent these through a Bio-Rad scan head, collecting second-harmonic generation in the forward and backward directions using Hamamatsu photomultiplier tubes. To improve the signal-to-noise ratio, they lowered the illumination intensity and collected data in a photon-counting mode for as long as 60 s.
Schematic of the second-harmonic-generation and multiphoton microscopy setup. This figure is adopted from Dombeck et al, PNAS, 2003.
They used the same setup, sans the photon counting, for multiphoton and fluorescence imaging. They did this on transgenic mice that expressed yellow fluorescent protein in neurons exclusively. This allowed them to more accurately identify the types of neurites containing polarized microtubule arrays.
Webb noted that the brain tissue preparations required for this study were demanding. They were, though, just as necessary for success as the imaging technology.
When the group investigated cultured neuronal cells, it found polarized microtubules in axons, with little polarization in mature dendrites. This outcome agreed with earlier work.
However, the situation was different in brain slices. There the researchers also found polarized microtubule arrays in mature dendrites of hippocampal and cortical-layer neurons. They observed the second-harmonic-generation signal from the apical dendrites of both labeled and unlabeled pyramidal neurons, suggesting that polarized microtubules were everywhere in such dendrites.
The ratio of forward- to backward-emitting second-harmonic signals (lower left) allowed an estimate of microtubule polarity in neuronal axons and dendrites (upper left and lower right). In cultured neurons, second-harmonic signals emitted by polarized microtubules match well with axons but not with dendrites. Courtesy of Alex C. Kwan and Watt W. Webb, Cornell University.
Their data showed that these organized arrays extended for distances greater than 270 μm, with a polarization – a common directionality – of more than 80 percent. They found that polarization was age-dependent, with young mice showing mixed polarity. The polarization increased with age until about 4 months.
As for why the microtubules might be aligned, one reason could be efficiency. Studies have predicted that transportation in a single direction is up to 10 times faster than bidirectional transportation for a 100-μm-long neurite, largely because the biochemical cargo spends more time actively moving.
These polarization results have implications for molecular trafficking. The various molecular motors that haul cargo around inside the cell preferably travel in one direction. So if the orientation of the microtubule array favors one polarization, one molecular motor would preferentially be used over its oppositely traveling counterpart.
Discovering whether that is the case is the subject of ongoing research, reported Webb. Although the second-harmonic-generation technique can reveal whether microtubules are oriented in common, it cannot uncover their direction. Molecular motor trafficking experiments done in conjunction with measurements of polarization could answer the question.
Another area of study involves transgenic mouse models that have neurodegenerative conditions such as Alzheimer’s disease. Being able to see if and how microtubule arrays are perturbed near lesions from such ailments could provide some crucial information, said Webb. “One prevalent hypothesis in Alzheimer’s disease is that the microtubule network is disrupted such that cargo transport is hindered and eventually leads to cell death.”
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